Abstract: Our overall goal is to identify the mechanisms through which TNF?-producing hematopoietic cells regulate blood vessels and perivascular HSPC (Hematopoietic stem and progenitor cells) vascular niches. HSPC are responsible for generating all the cells found in the blood and are indispensable for life. HSPC reside in the bone marrow associated with specific structures (niches) composed by different types of cells including endothelial cells, CD45-CD31-LepR+, CD45-CD31-Ng2+ cells and megakaryocytes. These niches maintain and regulate HSPC and are indispensable for HSC maintenance. However, little is known about the mechanisms that regulate the different components of the niche during homeostasis and regeneration. Identifying these mechanisms is critical to understand how HSPC are regulated in health and disease and because it might lead to novel therapies that target the niche to treat disease. In our preliminary studies we have discovered two entirely novel functions for TNF?-producing cells in hematopoiesis: 1) We found that bone marrow (but not peripheral) granulocytes use TNF? to create a regenerative microenvironment that promotes the expansion of the stromal niches that sustain hematopoiesis and accelerates hematopoietic recovery after autologous transplantation in mice. Our results suggest that manipulation of granulocytes and/or TNF? signals can be used to accelerate donor cell engraftment after transplantation. We also found, in human beta thalassemia patients, that higher numbers of granulocytes in the initial graft predicted faster peripheral and neutrophil engraftment. This data supports the hypothesis that granulocytes might also play a role in driving regeneration in human patients. 2) We found that TNF?- producing cells control the size of the perivascular niche and HSC localization to BM niches. Our results demonstrate that, during homeostasis, TNF?-producing cells regulate vascular niche abundance by controlling the numbers of endothelial cells and perivascular cells in the niche. We also found that, in Tnf?-/- mice, HSC relocate away from perivascular niches. In this proposal we investigate the mechanisms of this TNF?-mediated cellular crosstalk. In Aim 1 we will investigate the mechanisms through which granulocytes drive vascular and hematopoietic regeneration, their function in vascular homeostasis, and whether pharmacological manipulation of these granulocytes can be utilized to promote regeneration after myeloablation. In Aim 2 of this proposal we will utilize chimeric mice and genetic models for Tnfrsf1a (one of the TNF? receptors) reactivation and Tnf? deletion and a 5-color immunofluorescence imaging of HSC and their niches to identify the source and targets of TNF? that regulate HSC function and localization to the niche.